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Chromatography,
Thin Layer and Gas
- #Chromatography: An analytical technique that separates components
in a mixture between a mobile phase and stationary phase
- Chromatography is used in the analysis of drugs, pesticides and forensic evidence. Useful as it only requires
small samples. During chromatography a mobile phase sweeps a mixture over a stationary phase.
- #Phase: A physically distinctive form of a substance, such
as the solid, liquid and gaseous states of ordinary matter
- #Mobile Phase: The phase that
moves in chromatography
- #Stationary Phase: The phase that
does not move in chromatography
- Thin Layer Chromatography (TLC)
- The stationary phase is a solid, thin layer of an absorbent material e.g. silica gel (SiO2) or Alumina (AlO3). Coated on a flat inert support,
usually a sheet of glass of plastic, called the TLC plate. The mobile phase is a liquid solvent which moves vertically up the TLC plate.
- #Adsorption: The process by which a solid hold molecules of a gas, liquid
or solute as a thin film on the surface of a solid or, more rarely, a liquid
- Particularly useful for organic chemistry, it is quick
and simple and used to measure the extent of a
chemical reaction or check the purity of compunds
- A small sample of the mixture is dissolved and a small spot of it is placed a short distance from one end of the TLC plate. This is then allowed to dry and the TLC plate is a jar
containing a shallow layer of solvent. The solvent level must be below the level of the sample spot or it will be washed of the plate. The jar is then sealed to saturate the space
inside with solvent vapour, preventing solvent loss by evaporation. As the solvent rises up the TLC plate it meets the sample and the components in it are swept upwards.
- Separation is achieved by the adsorption of the components in the mixture to the surface of the solid stationary phase. Some will bind
more strongly and others more weakly. Resulting in different compounds being separated as they travel different distances up the plate.
To achieve maximum separation the solvent is left to rise until it almost reaches the top of the TLC plate. It is then taken out of the
solvent and the solvent front is marked with a pencil line, the solvent is allowed to evaporate and the result is the chromatogram.
- Each component appears as a spot on the chromatogram. If they are colourless they may need a
locating agent to 'develop' the chromatogram e.g. UV radiation to show them up by fluorescence
- Each component appears as a spot on the chromatogram. If they are colourless they may need a
locating agent to 'develop' the chromatogram e.g. UV radiation to show them up by fluorescence
- Separation is achieved by the adsorption of the components in the mixture to the surface of the solid stationary phase. Some will bind
more strongly and others more weakly. Resulting in different compounds being separated as they travel different distances up the plate.
To achieve maximum separation the solvent is left to rise until it almost reaches the top of the TLC plate. It is then taken out of the
solvent and the solvent front is marked with a pencil line, the solvent is allowed to evaporate and the result is the chromatogram.
- Rf Values
- Calculated by dividing the distance moved by the
component by the distance moved by the solvent front
- The value produced is between zero and one.
- The value produced is between zero and one.
- Can be a compared to a database of Rf values for pure
samples to identify an unknown substance.
- Calculated by dividing the distance moved by the
component by the distance moved by the solvent front
- Limitations
- Similar compounds often have similar Rf values making identification difficult.
- A completely unknown
compound has no reference
for comparison
- Trial and error may be required before a suitable solvent, or mixture of solvents, is discovered. If the components are very soluble
in a substance, they will just be washed up the TLC plate with the solvent front, if they have little solubility they will barely move.
- Similar compounds often have similar Rf values making identification difficult.
- The stationary phase is a solid, thin layer of an absorbent material e.g. silica gel (SiO2) or Alumina (AlO3). Coated on a flat inert support,
usually a sheet of glass of plastic, called the TLC plate. The mobile phase is a liquid solvent which moves vertically up the TLC plate.
- Gas Chromatography (GC)
- Takes place in a gas chromatograph. The stationary phase is a liquid, often a long chained alkane with a high boiling point or a solid e.g. a
silicone polymer. This is coated on the inside of capillary tubing, called a chromatography column, which is wound into a coil that fits into
thermostatically controlled oven. For separation of different types of compounds, columns containing different liquids and solids are used. The
mobile phase is a carrier gas which moves through the column, an inert or unreactive gas is usually such as helium or nitrogen is usually used.
- Used to separate volatile components in a mixture so is
useful for many organic compounds with low boiling points
- The mixture is injected into the gas chromatograph, where it is vaporised. The mobile carrier gas then flushes this through the
column. If the lining/stationary phase is a solid then the components may become adsorbed to the surface, if it is a liquid they may
dissolve. The greater solubility or adsorption, the more the components are slowed down as they move through the column causing
them to separate. Separation can be improved by using different oven temperatures and different flow rates for the carrier gas.
- Each component leaves the column at a different time and is detected as it leaves.
A computer displays the results as a gas chromatogram. The area under each
peak is proportional to the amount of a compound in the sample
- Each component leaves the column at a different time and is detected as it leaves.
A computer displays the results as a gas chromatogram. The area under each
peak is proportional to the amount of a compound in the sample
- #Retention Time: The time taken for a component to pass
from the column inlet to the detector in gas chromatography.
- Different compounds have different retention times, these can be compared
to a database of known compounds to identify an unknown compound.
- Limitations: Does not positively identify most compounds, as thousands of compounds could
have the same retention time, peak shape and so detector response. Substances may be
hidden behind others with a higher concentration and the same retention time and so not all
substances in a sample will be necessarily separated and detected. Unknown compounds
have no reference retention time for comparison.
- GC results are often used in court and so to decrease doubts about
the scientific method it is used combined with mass spectrometry.
- Takes place in a gas chromatograph. The stationary phase is a liquid, often a long chained alkane with a high boiling point or a solid e.g. a
silicone polymer. This is coated on the inside of capillary tubing, called a chromatography column, which is wound into a coil that fits into
thermostatically controlled oven. For separation of different types of compounds, columns containing different liquids and solids are used. The
mobile phase is a carrier gas which moves through the column, an inert or unreactive gas is usually such as helium or nitrogen is usually used.
- #Chromatogram: A visible record showing the separation showing the
result of separation of the components of a mixture by chromatography.
- Gas Chromatography - Mass Spectrometry
(GC-MS)
- GC separates components in the mixture, MS analysis
each in turn providing a mass spectrum.
- The is provides a more powerful analytical tool. MS provides a unique spectrum
for each compound that comes out of the GC allowing a positive identification
- Uses
- Forensics: Can analyse and identify
minute particles at a crime scene
- Environmental Analysis: Analysing and monitoring environmental pollutants in the environment.
e.g. quality of waste and drinking water, detecting pesticides in foods
- Airport Security: To detect explosives or traces
of explosives on luggage and on people
- Space Probes: Analyse the surface of mars and titan, and the atmosphere or venus
- Forensics: Can analyse and identify
minute particles at a crime scene
- GC separates components in the mixture, MS analysis
each in turn providing a mass spectrum.
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